System and Method of Modular Cloning

Abstract

System for producing a nucleic acid construct of interest, said system comprising:a set of n entry DNAs numbered 1 to n, n being an integer of at least 2,each of said n entry DNAs comprising in this order:(i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and(iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively;the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n;said system further comprising a destination vector comprising in this order:(I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);(III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and(IV) optionally, an insert between the recognition sites of item (III) and item (I);said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.

1 . System for producing a nucleic acid construct of interest, said system comprising:
 a set of n destination vectors (“destination vectors M”), n being an integer of at least 2, preferably at least 3,   each of said n destination vectors M comprising in the following order:   (I′) a type IIs restriction endonuclease recognition site defining the cleavage site of item (II′);   (II′) the cleavage site of said recognition site of item (I′);   (III′) a cleavage site of said recognition site of the following item (IV′);   (IV′) a further type IIs restriction endonuclease recognition site defining the cleavage site of item (III′) and being a different recognition site of a type IIs restriction endonuclease from that of item (I′);   (V′) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of item and   (IV′) and the following item (VI′);   (VI′) a further type IIs restriction endonuclease cleavage site;   (VII′) a type IIs restriction endonuclease recognition site of the cleavage site of item (VI′) and   (VIII′) optionally, an insert between the recognition sites of item (VII′) and item (I′); and   a set of n linkers M, n being as defined above, each linker M comprising in the following order:   (a′) a type IIs restriction endonuclease recognition site defining the cleavage site of item (b′);   (b′) the cleavage site of said recognition site of item (a′);   (c′) a cleavage site of a further type IIs restriction endonuclease recognition site of item (d′), said cleavage site having the same sequence of nucleotides as the cleavage site of item (b′);   (d′) the type IIs restriction endonuclease recognition site defining the cleavage site of item (c′) and being a different recognition site of a type IIs restriction endonuclease different from that of item (a′);   (e′) a further cleavage site of a type IIs restriction endonuclease recognition site of the following item (f′);   (f′) the type IIs restriction endonuclease recognition site defining the cleavage site of item (e′), that is preferably a recognition site of the same endonuclease as the recognition site of item (a′);   wherein   the cleavage sites (VI′) of all n destination vectors M are identical;   the cleavage sites (e′) of all n linkers M are identical;   the cleavage site of item (VI′) of each destination vector M is complementary to the cleavage site of item (e′) of each linker M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites (VII′) and (f′);   the cleavage sites of items (II′) and (III′) within each destination vector M have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (II′) and that of item (III′); and   the cleavage sites of items (b′) and (c′) within each linker M have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (b′) and that of item (c′); and   the cleavage site (II′) of each destination vector M is unique among the cleavage sites (II′) of the set of n destination vectors M such that there are n different cleavage sites (II′), wherein for each of said n different cleavage sites (II′), there is a linker M having a cleavage site (b′) of identical nucleotide sequence among the set of n linkers M.   
     
     
         2 . The system according to  claim 1 , wherein
 (α) the recognition sites of items (a′) and (f′) of all n linkers M are recognition sites of the same type IIs restriction endonuclease;   (β) the recognition sites of items (d′) of all n linkers M are recognition sites of the same type IIs restriction endonuclease;   wherein the recognition sites of item (a) are different recognitions sites from those of item (β).   
     
     
         3 . The system according to  claim 1 , wherein
 (γ) the recognition sites of items (I′) and (VII′) of all n destination vectors M are recognition sites of the same type IIs restriction endonuclease;   (δ) the recognition sites of items (IV′) of all n destination vectors M are recognition sites of the same type IIs restriction endonuclease;   wherein the recognition sites of item (γ) are different recognitions sites from those of item (δ).   
     
     
         4 . The system according to  claim 1 , wherein
 the recognition sites of items (VII′) and (I′) of destination vectors M and of items (a′) and (f′) of the linkers M are recognition sites of the same type IIs restriction endonuclease;   the recognition sites of items (IV′) of destination vectors M and of items (d′) of the linkers M are recognition sites of the same type IIs restriction endonuclease.   
     
     
         5 . The system according to  claim 1 , comprising:
 a set of z entry DNAs numbered 1 to z, z being an integer of at least 2, preferably an integer of at least 3,   each of said z entry DNAs comprising in this order:   (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and   (iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;   wherein   the cleavage site of item (i) of each entry DNA is complementary to the cleavage site of item (II′) of one of the n destination vectors M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites of items (i) and (I′),   the recognition sites of item (i) of all z entry DNAs are preferably recognition sites of the same type IIs restriction endonuclease as the recognition sites of item (I′) and (VII′);   the cleavage site of item (iii) of each entry DNA is complementary to the cleavage sites of item (b′) of one of the n linkers M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites of items (iii) and (a′),   the recognition sites of item (i) are recognition sites of the same type IIs restriction endonuclease as the recognition sites of item (a′) and (f′); and   the recognition sites of items (i) and (iii) of all z entry DNAs are recognition sites of the same type IIs restriction endonuclease.   
     
     
         6 . The system according to  claim 1 , further comprising a set of n destination vectors (“destination vectors P”), wherein n is as defined in  claim 1 ,
 each of said n destination vectors P comprising in the following order: 
 (I″) a type IIs restriction endonuclease recognition site defining the cleavage site of item (II″); 
 (II″) the cleavage site of said recognition site of item (I″); 
 (III″) a cleavage site of said recognition site of the following item (IV″); 
 (IV″) a further type IIs restriction endonuclease recognition site defining the cleavage site of item (III″) and being a different recognition site of a type IIs restriction endonuclease from that of item (I″); 
 (V″) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of item and (IV″) and the following item (VI″); 
 (VI″) a further type IIs restriction endonuclease cleavage site; 
 (VII″) a type IIs restriction endonuclease recognition site of the cleavage site of item (VI″), preferably of the same endonuclease as the recognition site of item (I″) and 
 (VIII″) optionally, an insert between the recognition sites of item (VII″) and item (I″); and 
 a set of n linkers P, each linker P comprising in the following order: 
 (a″) a type IIs restriction endonuclease recognition site defining the cleavage site of item (b″); 
 (b″) the cleavage site of said recognition site of item (a″); 
 (c″) a cleavage site of a further type IIs restriction endonuclease recognition site of item (d″), said cleavage site having the same nucleotide sequence as the cleavage site of item (b″); 
 (d″) the type IIs restriction endonuclease recognition site defining the cleavage site of item (c″) and being a different recognition site of a type IIs restriction endonuclease from that of item (a″); 
 (e″) a further cleavage site of a type IIs restriction endonuclease recognition site of the following item (f″); 
 (f″) the type IIs restriction endonuclease recognition site defining the cleavage site of item (e″), that is preferably a recognition site of the same endonuclease as the recognition site of item (a″); 
 wherein 
 the cleavage sites (VI″) of all n destination vectors P are identical; 
 the cleavage sites (e″) of all n linkers P are identical; 
 the cleavage site of item (VI″) of each destination vector P is complementary to the cleavage site of item (e″) of each linker P for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites (VII″) and (f″); 
 the cleavage sites of items (II″) and (III″) within each destination vector P have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of item (II″) and the cleavage site of item (III″); 
 the cleavage sites of items (b″) and (c″) within each linker P have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (b″) and the cleavage site of item (c″); and 
 for each of said n different cleavage sites (b′) or (II′), there is a destination vector P having a cleavage site (II″) of identical nucleotide sequence as the nucleotide sequence of cleavage sites (b′) or (II′); and 
 for each of said n different cleavage sites (b′) or (II′), there is a linker P having a cleavage site (b″) of identical nucleotide sequence as the nucleotide sequence of cleavage sites (b′) or (II′). 
 
     
     
         7 . The system according to  claim 6 , wherein
 the recognition sites of items (I″) and (VII″) of all n destination vectors P are recognition sites of the same type IIs restriction endonuclease; and   the recognition sites of items (IV″) of all n destination vectors P are recognition sites of the same type IIs restriction endonuclease but different from the recognition sites of items (I″) and (VII″).   
     
     
         8 . The system according to  claim 6 , wherein
 the recognition sites of items (I″), (IV′), (d′), (a″) and (f″) are recognition sites of the same type IIs restriction endonuclease;   the recognition sites of items (IV″), (I′), (VII′), (a′) and (f′) are recognition sites of the same type IIs restriction endonuclease.   
     
     
         9 . Use of the system defined in  claim 1  for cloning a nucleic acid construct of interest into a vector.